Use of etodolac for the treatment of chronic lymphocytic leukemia

ABSTRACT

A method of treating cancer is provided comprising administering an amount of etodolac to a subject afflicted with cancer that is effective to reduce the viability and/or to sensitize the cancer to an anti-cancer agent.

The invention was made with Government support under Grant No. GM23200awarded by the National Institute of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Chronic lymphocytic leukemia (CLL) is the most common leukemia in theUnited States. CLL involves the cancerous proliferation of lymphocytes.It is most common among older adults; 90 percent of the cases are inpeople more than 50 years old. It occurs 1-3 times more often among menthan among women. The onset of CLL tends to be insidious, with symptomsdeveloping gradually. Because it involves an overproduction of mature,functional lymphocytes, persons with this disorder may survive foryears. In contrast, in some, the disorder proceeds very rapidly, andrequires immediate treatment. Currently, the adenine deoxynucleosidesfludarabine (fludara) and 2-chlorodeoxyadenosine (2CdA) are the drugs ofchoice to treat the disease. However, clinical remissions are seldominduced, and patients eventually succumb from their leukemia.

The number of nonsteroidal anti-inflammatory drugs (NSAIDs) hasincreased to the point where they warrant separate classification. Inaddition to aspirin, the NSAIDs available in the U.S. includemeclofenamate sodium, oxyphenbutazone, phenylbutazone, indomethacin,piroxicam, sulindac and tolmetin for the treatment of arthritis;mefenamic acid and zomepirac for analgesia; and ibuprofen, fenoprofenand naproxen for both analgesia and arthritis. Ibuprofen, mefenamic acidand naproxen are used also for the management of dysmenorrhea.

The clinical usefulness of NSAIDs is restricted by a number of adverseeffects. Phenylbutazone has been implicated in hepatic necrosis andgranulomatous hepatitis; and sulindac, indomethacin, ibuprofen andnaproxen with hepatitis and cholestatic hepatitis. Transient increasesin serum aminotransferases, especially alanine aminotransferase, havebeen reported. All of these drugs, including aspirin, inhibitcyclooxygenase, that in turn inhibits synthesis of prostaglandins, whichhelp regulate glomerular filtration and renal sodium and waterexcretion. Thus, the NSAIDs can cause fluid retention and decreasesodium excretion, followed by hyperkalemia, oliguria and anuria.Moreover, all of these drugs can cause peptic ulceration. See,Remington's Pharmaceutical Sciences, Mack Pub. Co., Easton, Pa. (18thed., 1990) at pages 1115-1122.

There is a large amount of literature on the effect of NSAIDs on cancer,particularly colon cancer. For example, see H. A. Weiss et al., Scand J.Gastroent., 31, 137 (1996) (suppl. 220) and Shiff et al., Exp. CellRes., 222, 179 (1996). More recently, B. Bellosillo et al., Blood, 92,1406 (1998) reported that aspirin and salicylate reduced the viabilityof B-cell CLL cells in vitro, but that indomethacin, ketoralac andNS-398, did not.

C. P. Duffy et al., Eur. J. Cancer, 34, 1250 (1998), reported that thecytotoxicity of certain chemotherapeutic drugs was enhanced when theywere combined with certain non-steroidal anti-inflammatory agents. Theeffects observed against human lung cancer cells and human leukemiacells were highly specific and not predictable; i.e., some combinationsof NSAID and agent were effective and some were not. The only conclusiondrawn was that the effect was not due to the cyclooxygenase inhibitoryactivity of the NSAID.

The Duffy group filed a PCT application (WO98/18490) on Oct. 24, 1997,directed to a combination of a “substrate for MRP”, which can be ananti-cancer drug, and a NSAID that increases the potency of theanti-cancer drug. NSAIDs recited by the claims are acemetacin,indomethacin, sulindac, sulindac sulfide, sulindac sulfone, tolmetin andzomepirac. Naproxen and piroxicam were reported to be inactive.

Therefore, a continuing need exists for methods to control cancers, suchas leukemias, and to increase the potency of anti-cancer drugs withrelatively non-toxic agents.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a therapeutic method totreat leukemia, e.g. chronic lymphocytic leukemia, comprisingadministering to a mammal afflicted with leukemia an amount of etodolac,R(−) etodolac, or an analog thereof, effective to inhibit the viabilityof leukemic cells of said mammal. The present invention also provides amethod of increasing the susceptibility of human leukemia cells, such aschronic lymphocytic leukemia (CLL) cells, to a chemotherapeutic agentcomprising contacting the cells with an effective sensitizing amount ofetodolac, or an analog thereof. Thus, the invention provides atherapeutic method for the treatment of a human or other mammalafflicted with a leukemia such as CLL, wherein an effective amount ofetodolac or an analog thereof is administered to a subject afflictedwith leukemia and undergoing treatment with a chemotherapeutic(“antineoplastic”) agent.

Preferably, the R(−) isomer of etodolac is administered in conjunctionwith one or more chemotherapeutic agents effective against CLL such asfludarabine (fludara) or 2-chlorodeoxyadenosine (2CdA). Unexpectedly,the R(−) isomer of etodolac, which exhibits little anti-inflammatoryactivity, was found to be responsible for the sensitizing activity ofracemic etodolac. Therefore, the present invention also provides amethod to treat other forms of cancer, such as breast, prostate andcolon cancer with RS or the R(−) enantiomer of etodolac or an analogthereof, used alone, or preferably, in combination with achemotherapeutic agent.

A method of evaluating the ability of etodolac to sensitize leukemiacells, such as CLL cells, to a chemotherapeutic agent is also provided.The assay method comprises (a) isolating a first portion of leukemiacells, such as leukemic B cells, from the blood of a human leukemiapatient; (b) measuring their viability; (c) administering etodolac, oran analog thereof, to said patient; (d) isolating a second portion ofleukemia cells from said patient; (e) measuring the viability of thesecond portion of leukemia cells; and (f) comparing the viabilitymeasured in step (e) with the viability measured in step (b); whereinreduced viability in step (e) indicates that the cells have beensensitized to said chemotherapeutic agent.

Preferably, steps (b) and (e) are carried out in the presence of thechemotherapeutic agent, as will be the case when the leukemia cells arederived from the blood of a mammal afflicted with leukemia, such as aCLL patient.

Thus, a cancer patient about to undergo, or undergoing, treatment forleukemia can be rapidly evaluated to see if he/she will benefit fromconcurrent chemotherapy and administration of etodolac or an analogthereof.

The present invention is based on the discovery by the inventors thatracemic etodolac inhibits the viability of purified CLL cells atconcentrations that do not inhibit the viability of normal peripheralblood lymphocytes (PBLs). It was then unexpectedly found that the R(−)enantiomer of etodolac is as toxic to CLL cells as is the S(+)enantiomer. It was then found that etodolac synergistically interactedwith fludarabine and 2-chloroadenosine to kill CLL cells atconcentration at which the chemotherapeutic agents alone were inactive.

Etodolac and its analogs possess several unique disposition features dueto their stereoselective pharmacokinetics. In plasma, after theadministration of RS-etodolac, the concentrations of the “inactive”R-enantiomer of etodolac are about 10-fold higher than those of theactive S-enantiomer, an observation that is novel among the chiralNSAIDs. See, D. R. Brocks et al., Clin. Pharmacokinet., 26, 259 (1994).After a 200 mg dose in six elderly patients, the maximum plasmaconcentration of the R-enantiomer was about 33 μM. In contrast, themaximum concentration of the S-enantiomer was 5-fold lower. The typicaldosage of the racemic mixture of etodolac is 400 mg BID, and the drughas an elimination half-life between 6-8 hours. Thus, etodolac atcommonly used dosages should achieve a plasma concentration of theR-enantiomer shown to sensitize CLL cells in vitro to fludarabine.Moreover, it is likely that the administration of the purifiedR-enantiomer will not display the side effects associated withcyclooxygenase (COX) inhibitors, such as ulcers and renal insufficiency,and thus could be given at considerably higher dosages. It is believedthat etodolac can act both directly and indirectly against cancer cells;i.e., by inhibiting factor(s) that would normally block apoptosis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph depicting the sensitivity of normal peripheral bloodlymphocytes (PBL) to racemic etodolac.

FIG. 2 is a graph depicting the sensitivity of CLL cells to racemicetodolac.

FIG. 3 is a graph depicting the synergistic effect of a combination ofracemic etodolac and fludarabine against CLL cells.

FIG. 4 is a graph depicting the synergistic effect of a combination of50 μM etodolac with 10 μM 2CdA or 10 mM Fludara against CLL cells.

FIG. 5 is a graph depicting the sensitivity of CLL cells to S- andR-etodolac.

FIGS. 6 and 7 depict the viability of CLL cells from two patients beforeand after etodolac administration.

FIG. 8 depicts a flow cytometric analysis of CLL cells before and afteretodolac treatment.

DETAILED DESCRIPTION OF THE INVENTION

Etodolac (1,8-diethyl-1,3,4,9-tetrahydro[3,4-6]indole-1-acetic acid) isa NSAID of the pyranocarboxylic acid class, that was developed in theearly 1970s. See, C. A. Demerson et al., Ger. Pat. No. 2,226,340 (Am.Home Products); R. R. Martel et al., Can. J. Pharmacol., 54, 245 (1976).Its structure is formula (I), below:wherein (*) denotes the chiralcenter. See also, The Merck Index, (11th ed.), at page 608.

Early studies indicated that etodolac was an effective NSAID, with afavorable ratio of anti-inflammatory activity to adverse effects.Etodolac has been marketed for some years in Europe, including the UK,Italy, France and Switzerland, for the treatment of pain andinflammation associated with various forms of arthritis. The drug wasmore recently given approval for marketing in the U.S., where itsapproved uses are currently restricted to the treatment ofosteoarthritis, and as a general purpose analgesic.

The pharmacokinetics of etodolac have been extensively reviewed by D. R.Brocks et al., Clin. Pharmacokinet., 26, 259 (1994). Etodolac ismarketed as the racemate. However, Demerson et al., J. Med. Chem., 26,1778 (1983) found that the (+)-enantiomer of etodolac possessed almostall of the anti-inflammatory activity of the racemate, as measured byreduction in paw volume of rats with adjuvant polyarthritis, andprostaglandin synthetase inhibitory activity of the drug. No activitywas discernible with the (−)-enantiomer. The absolute configurations ofthe enantiomers were found to be S-(+) and R-(−), which is similar tothat for most other NSAIDs. The enantiomer of the racemate can beresolved by fractional crystallization using optically active1-phenylethylamine, and use of HPLC to determine racemic etodolac andenantiomeric ratios of etodolac and two hydroxylated metabolites hasbeen reported by U. Becker-Scharfenkamp et al., J. Chromatog., 621, 199(1993) and B. M. Adger et al. (U.S. Pat. No. 5,811,558). The in vivodisposition of etodolac is extremely stereoselective, with plasmaconcentrations of the “inactive” R-enantiomer greatly exceeding those ofthe “active” S-enantiomer. In this respect, etodolac is unique inrelation to other chiral NSAIDs, for which the “active” S-enantiomerusually attains plasma concentrations that are similar to or higher thanthose of the “inactive” enantiomer. Nonetheless, the R(−) enantiomer hasbeen asserted to have some analgesic activity. See, Young et al., U.S.Pat. No. 5,561,151. However, as exemplified below, this unusualdisposition facilitates administration of amounts of etodolac that areeffective to sensitize leukemic cells to chemotherapeutic agents,without giving rise to the side effects of the “active”anti-inflammatory S(+) enantiomer.

The chemical synthesis of the racemic mixture of etodolac can beperformed by the method described in Demerson et al., U.S. Pat. No.3,843,681; and C. A. Demerson et al., J. Med. Chem., 19(3), 391 (1976),the disclosures of which are hereby incorporated by reference.

The R(−) isomer of etodolac may be obtained by resolution of the mixtureof enantiomers of etodolac using conventional means, such as theformation of a diastereomeric salt with a basic optically activeresolving acid; see, for example, “Stereochemistry of Carbon Compounds,”by E. L. Eliel (McGraw Hill, 1962); C. H. Lochmuller et al., JChromatog., 113, 283 (1975); “Enantiomers, Racemates and Resolutions,”by J. Jacques, A. Collet, and S. H. Wilen, (Wiley-Interscience, NewYork, 1981); and S. H. Wilen, A. Collet, and J. Jacques, Tetrahedron,33, 2725 (1977).

Analogs of etodolac that can be used in the present inventions aredisclosed inter alia, in C. A. Demerson (U.S. Pat. No. 3,843,681), ascompounds of formula (II):

in which R¹ is selected from the group consisting of lower alkyl, loweralkenyl, lower alkynyl, lower cycloalkyl, phenyl, benzyl and 2-thienyl,R², R³, R⁴ and R⁵ are the same or different and are each selected fromthe group consisting of hydrogen and lower alkyl, R⁶ is selected fromthe group consisting of hydrogen, lower alkyl, hydroxy, lower alkoxy,benzyloxy, lower alkanoyloxy, nitro and halo, R⁷ is selected from thegroup consisting of hydrogen, lower alkyl and lower alkenyl, X isselected from the group consisting of oxy and thio, Y is selected fromthe group consisting of carbonyl or (C₁-C₃)alkylC(O), wherein each alkylis substituted with O-2 (C₁-C₄)alkyl and Z is selected from the groupconsisting of hydroxy, lower alkoxy, amino, lower alkylamino,di(lower)alkylamino and phenylamino, or a pharmaceutically acceptablesalt thereof. Lower (alkyl, alkenyl, alkanoyl, etc.) indicates a C₁-C₆group, preferably a C₁-C₄ group.

The magnitude of a prophylactic or therapeutic dose of racemic orR-etodolac in the acute or chronic management of cancer, i.e., CLL, willvary with the stage of the cancer, such as the solid turmor or leukemiato be treated, the chemotherapeutic agent(s) or other anti-cancertherapy used, and the route of administration. The dose, and perhaps thedose frequency, will also vary according to the age, body weight, andresponse of the individual patient. In general, the total daily doserange for racemic or R-etodolac, for the conditions described herein, isfrom about 50 mg to about 5000 mg, in single or divided doses.Preferably, a daily dose range should be about 100 mg to about 2500 mg,in single or divided doses. In managing the patient, the therapy shouldbe initiated at a lower dose and increased depending on the patient'sglobal response. It is further recommended that infants, children,patients over 65 years, and those with impaired renal or hepaticfunction initially receive lower doses, and that they be titrated basedon global response and blood level. It may be necessary to use dosagesoutside these ranges in some cases. Further, it is noted that theclinician or treating physician will know how and when to interrupt,adjust or terminate therapy in conjunction with individual patientresponse. The terms “an effective amount” or “an effective sensitizingamount” are encompassed by the above-described dosage amounts and dosefrequency schedule.

Any suitable route of administration may be employed for providing thepatient with an effective dosage of etodolac, i.e., R(−)etodolac. Forexample, oral, rectal, parenteral (subcutaneous, intravenous,intramuscular), intrathecal, transdermal, and like forms ofadministration may be employed. Dosage forms include tablets, troches,dispersions, suspensions, solutions, capsules, patches, and the like.The etodolac may be administered prior to, concurrently with, or afteradministration of chemotherapy, or continuously, i.e., in daily doses,during all or part of, a chemotherapy regimen. The etodolac, in somecases, may be combined with the same carrier or vehicle used to deliverthe anti-cancer chemotherapeutic agent.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrated agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like a syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anon-toxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, non-toxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

The invention will be further described by reference to the followingdetailed examples.

EXAMPLE 1

Sensitivity of Normal Peripheral Blood Lymphocytes and CLL Cells toEtodolac.

Mononuclear cells were isolated from the peripheral blood of B-CLLpatients and normal donors using density gradient centrifugation(Ficoll-Paque). Cells were cultured at 2×10⁶ cells per mL in RPMI with20% autologous plasma in 96-well plates with or without the indicated μMconcentrations of etodolac (racemic, S-etodolac, R-etodolac) and incombination with 2-chloro-2′-deoxyadenosine (2CdA) or fludarabine. Atindicated times (12, 24, 36, 48, 60, 72 hours), viability assays wereperformed using the erythrocin B exclusion assay, as described by D.Carson et al., PNAS USA, 89, 2970 (1992).

As shown in FIG. 1, significant death of normal PBLs occurred only at800 μM racemic etodolac, a concentration which cannot be obtained invivo.

Peripheral blood lymphocytes from a normal donor were cultured with 1.0mM etodolac for 24 hours. Then B lymphocytes were identified by stainingwith anti-CD19 antibody, and viability was assessed by DiOC₆fluorescence. Etodolac under these conditions did not reduce theviability of the normal B cells, compared to control cultures. When thesame viability assay was run with purified CLL cells from the peripheralblood of a CLL patient, the results were different. As shown in FIG. 2,50% of the CLL cells were killed by a 48 hour exposure to 200 μM racemicetodolac. More than 95% of the treated cells were malignant Blymphocytes.

EXAMPLE 2

Synergistic Combinations of Etodolac and Chemotherapeutic Agents

Fludarabine is a nucleoside analog commonly used for the treatment ofCLL. In this experiment the in vitro survival of CLL cells at theindicated time points was compared in cultures containing medium alone(“Con”, squares), fludarabine 10 nM (diamonds), etodolac 10 μM (closedcircles), and fludarabine 10 nM plus etodolac 10 μM (open circles). Thetwo drugs together exhibited a synergistic cytotoxic effect. FIG. 3shows that the combination killed 50% of CLL cells during 48 hours ofculture, while either drug alone was ineffective. FIG. 4 demonstratessynergy between 50 μM etodolac and 10 nM 2-chlorodeoxyadenosine andfludarabine, under the same test conditions.

EXAMPLE 3

Effect of R(−) and S(+) Etodolac Against CLL Cells.

Etodolac tablets were ground in a mortar and extracted from theformulation using ethyl acetate. The resulting racemic mixture ofenantiomers was separated into R and S isomers on a preparative scale byfractional crystallization by the procedure of Becker-Scharfenkamp andBlaschke, J. Chromatog., 621, 199 (1993). Thus, the racemic mixturesolid was dissolved in absolute 2-propanol and S-1-phenylethylamine wasadded to the solution. The resulting salt solution was stored in therefrigerator for 4 days. The crystalline white salt product was filteredand washed with cold 2-propanol and recrystallized two more times from2-propanol. The same procedure was repeated for the R isomer only usingR-1-phenylethylamine as the resolving agent. Finally, the R and S saltswere decomposed using 10% sulfuric acid (v/v) and extracted with ethylacetate. The chiral purity of each isomer was verified by HPLC using aChiral-AGP column from ChromTech.

The toxicities of the two enantiomers to CLL cells cultured in RPMI 1640medium with 10% autologous plasma were compared at the indicatedconcentrations and time points, as shown in FIG. 5. The R- andS-enantiomers are equivalently cytotoxic to the CLL cells.

EXAMPLE 4

Viability of CLL Cells Before and After Etodolac Treatment.

Heparanized blood was taken from two patients (JK and NA) with CLL. Theneach patient immediately took a 400 mg etodolac tablet, and a secondtablet 12 hours later. After another 12 hours, a second blood specimenwas obtained. The CLL cells were isolated and their survival in vitrowere compared in RPMI 1640 medium containing 10% autologous plasma, asdescribed in Example 1. The circles show CLL cells before etodolactreatment. In FIGS. 6-7, the upward pointing triangles represent CLLcell viability after etodolac treatment, wherein the cells are dispersedin medium containing the pretreatment plasma. The downward pointingtriangles are CLL cells after treatment maintained in medium with thepost-treatment plasma.

FIG. 6 shows the different survivals of the two cell populations frompatient J K. Note that the cells after treatment had a shortenedsurvival compared to the cells before treatment. FIG. 7 shows a lessdramatic but similar effect with patient N A. FIG. 8 is a flowcytometric analysis of CLL cells from patient J K before and afteretodolac treatment. DiOC₆ is a dye that is captured by mitochondria.When cells die by apoptosis, the intensity of staining decreases. The Xaxis on the four panels in FIG. 8 shows the DiOC₆ staining. An increasednumber of dots in the left lower box indicates cell death by apoptosis.If one compares the cells taken from the patient before etodolactreatment, and after etodolac treatment, one can see that the number ofdots in the left lower box is much higher after the drug. This effect isdetectable at 12 hours, and increases further after 24 hours.

To conduct the flow cytometric analysis, the mitochondrial transmembranepotential was analyzed by 3,3′ dihexyloxacarboncyanide iodide (DiOC₆),cell membrane permeability by propidium iodide (PI)³ and mitochondrialrespiration by dihydrorhodamine 123 (DHR) (See J. A. Royall et al.,Arch. Biochem. Biophys., 302, 348 (1993)). After CLL cells were culturedfor 12 or 24 hours with the indicated amount of etodolac, the cells wereincubated for 10 minutes at 37° C. in culture medium containing 40 nM ofDiOC₆ and 5 μg/ml PI. Cells were also cultured for 3 hours with theindicated amount of etodolac, spun down at 200×g for 10 minutes andresuspended in fresh respiration buffer (250 mM sucrose, 1 g/L bovineserum albumin, 10 mM MgCl₂, 10 mM K/Hepes, 5 mM KH₂PO₄ (pH 7.4)) andcultured for 10 minutes at 37° C. with 0.04% digitonin. Then cells wereloaded for 5 minutes with 0.1 μM dihydrorhodamine (DHR). Cells wereanalyzed within 30 minutes in a Becton Dickinson FAC-Scaliburcytofluorometer. After suitable comprehension, fluorescence was recordedat different wavelength: DiOC₆ and DHR at 525 nm (Fl-1) and PI at 600 nm(FL-3).

As a general matter a reduction of 10% in the survival of thepost-treatment malignant cells, compared to the pretreatment malignantcells, at 16 hours after culture in vitro is considered a “positive” inthis test, and indicates the use of etodolac, i.e., R(−) etodolac in CLLor other cancer therapy.

All of the publications and patent documents cited hereinabove areincorporated by reference herein. The invention has been described withreference to various specific and preferred embodiments and techniques.However, it should be understood that many variations and modificationsmay be made while remaining within the spirit and scope of theinvention.

What is claimed is:
 1. A method of the reducing the viability of human leukemia cells sensitive to a 1-(R) compound of formula (I):

wherein R¹ is lower alkyl, lower alkenyl, lower alkynyl, lower cycloalkyl, phenyl or benzyl, R², R³, R⁴ and R⁵ are the same or different and are each hydrogen or lower alkyl; R⁶ is hydrogen, lower alkyl, hydroxy, lower alkoxy, benzyloxy, lower alkanoyloxy, nitro or halo, R⁷ is hydrogen, lower alkyl or lower alkenyl; X is oxy; Y is carbonyl or (C₁-C₃)alkyl(CO), wherein each alkyl is substituted with 0-2 (C_(1-C) ₄) alkyl, and Z is hydroxy, lower alkoxy, amino, lower alkylamino, di(lower)alkylamino or phenylamino, or a pharmaceutically acceptable salt thereof, comprising administering an effective amount of the (R)-compound of formula (I) or the salt thereof to a human cancer patient afflicted with a leukemia and wherein R compound is substantially free of its S(+) stereoisomer.
 2. A method comprising the killing of human leukemia cells sensitive to a 1-(R) compound of formula (I):

wherein R¹ is lower alkyl, lower alkenyl, lower alkynyl, lower cycloalkyl, phenyl or benzyl, R², R³, R⁴ and R⁵ are the same or different and are each hydrogen or lower alkyl; R⁶ is hydrogen, lower alkyl, hydroxy, lower alkoxy, benzyloxy, lower alkanoyloxy, nitro or halo, R⁷ is hydrogen, lower alkyl or lower alkenyl; X is oxy; Y is carbonyl or (C₁-C₃)alkyl(CO), wherein each alkyl is substituted with 0-2 (C₁-C₄) alkyl, and Z is hydroxy, lower alkoxy, amino, lower alkylamino, di(lower)alkylamino or phenylamino, or a pharmaceutically acceptable salt thereof, comprising administering an effective amount of the compound of formula (I) or the salt thereof to a human cancer patient afflicted with a leukemia and wherein R compound is substantially free of its S(+) stereoisomer.
 3. The method of claim 1 or 2 wherein the leukemia is chronic lymphocytic leukemia.
 4. The method of claim 1 or 2 wherein the compound of formula (I) is R(−) etodolac.
 5. The method of claim 1 or 2 wherein the compound of formula (I) or the salt thereof is administered orally. 